Twoplex ^12/13C₆ aniline stable isotope and linkage‐specific sialic acid labeling 2D‐LC‐MS workflow for quantitative N‐glycomics

Abstract

Quantitative glycomics represents an actively expanding research field ranging from the discovery of disease-associated glycan alterations to the quantitative characterization of N-glycans on therapeutic proteins. Commonly used analytical platforms for comparative relative quantitation of complex glycan samples include MALDI-TOF-MS or chromatographic glycan profiling with subsequent data alignment and statistical evaluation. Limitations of such approaches include run-to-run technical variation and the potential introduction of subjectivity during data processing. Here, we introduce an offline 2D LC-MS workflow for the fractionation and relative quantitation of twoplex isotopically labeled N-linked oligosaccharides using neutral C and C aniline (Δmass = 6 Da). Additional linkage-specific derivatization of sialic acids using 4-(4,6-dimethoxy-1,3,5-trizain-2-yl)-4-methylmorpholinium chloride offered simultaneous and advanced in-depth structural characterization. The potential of the method was demonstrated for the differential analysis of structurally defined N-glycans released from serum proteins of patients diagnosed with various stages of colorectal cancer. The described twoplex C / C aniline 2D LC-MS platform is ideally suited for differential glycomic analysis of structurally complex N-glycan pools due to combination and analysis of samples in a single LC-MS injection and the associated minimization in technical variation.

Carbon Nanoparticles and Graphene Nanosheets as MALDI Matrices in Glycomics: a New Approach to Improve Glycan Profiling in Biological Samples

“…Among the different analytical methods that have been reported for glycans analysis, MS techniques coupled with a variety of dissociation techniques, including high- or low-energy collision-induced fragmentation (CID), post-source decay (PSD), infrared multiphoton dissociation (IRMPD), electron excitation dissociation (EED), electron capture dissociation (ECD), electron transfer dissociation (ETD) and electron detachment dissociation (EDD), has proven to be a powerful tools for glycan profiling [7–17]. In this regard, MALDI offers several advantages over other ionization techniques, mainly ease of operation, short analysis times, modest sample size requirements and relative simplicity of spectral interpretation [12, 18, 19].…”

Analysis of carbohydrates and glycoconjugates by matrix‐assisted laser desorption/ionization mass spectrometry: An update for 2017–2018

“…The mass difference of 32 Da allows a simple mass measurement to identify the linkage. The method has been adopted and improved by several investigators and is now a standard technique in this field (Albrecht et al, 2017; Snyder et al, 2017). Amides often replace esters and modifications of the lactones have improved their stability.…”

“…Compounds are usually quantified using analytes labelled with different stable isotopes such as normal isotopes in the derivative for the measured analyte and heavy isotopes (usually 2 H, 13 C, 15 N, or 18 O) in the reference material. Thus, for example, N ‐glycans have been measured by LC/MS using 12 / 13 C 6 aniline‐labelled compounds (Albrecht et al, 2017). Variations on the method include derivatives with different two or more stable isotopes such that their nominal masses are equal but which, upon fragmentation, yield fragments of different mass (see the six‐plex assay developed by Barrientos and Zhang (2018), described below).…”

gQuant, an Automated Tool for Quantitative Glycomic Data Analysis

“…Many efforts have been made to develop mass spectrometry (MS)-based glycan quantitation techniques due to the excellent qualitative ability, sensitivity and high throughput of mass spectrometry ( Wuhrer, 2013 ; Cao et al, 2020 ). For example, a metabolic labeling strategy was introduced for in vivo labeling and glycan quantitation ( Orlando et al, 2009 ); chemical labeling strategies using different isotope labeling reagents, such as 2-aminobenzoic acid, aniline, arginine, and 1-phenyl-3-methyl-5-pyrazolone, were developed for glycan derivatization and quantitation ( Prien et al, 2010 ; Albrecht et al, 2017 ; Cai et al, 2015 ; Smith et al, 2017 ; Wang et al, 2017 ); and enzymatic 18 O labeling was also utilized for glycan relative quantification ( Zhang et al, 2015 ; Cao et al, 2015 ). Among the developed techniques, MALDI-MS-based methods have shown high feasibility, efficiency and speed in quantitative glycan analysis and have been widely used.…”